Project Summary/Abstract Approximately 450,000 women succumb to breast cancer each year, making it the most common cause of female cancer mortality globally, with the majority of these deaths resulting from metastatic disease. In the treatment of metastases, the initial drug response rate is only about 50%, as compared to 90% observed in the treatment of primary tumors. Furthermore, resistance to these systemic therapies typically develops more quickly in the metastatic setting, thus emphasizing the critical need to improve our understanding of the mechanisms governing development of drug resistance. While chemotherapy effectively eliminates tumor cells, it has also been shown to simultaneously induce various counter-regulatory responses in the cells and tissues of the host, which leads to the development of a protective environment that promotes survival of few remaining tumor cells, often referred to as cancer stem cells (CSCs). Although numerous studies have focused on tumor cell intrinsic mechanisms of drug resistance, there have been very few investigations into extrinsic mechanisms of drug resistance mediated by these changes in the host cells and tissue environment. Periostin (POSTN) is a structural support protein found during normal tissue development, but whose expression has also been shown to be increased in cancer development and metastasis. During metastasis, POSTN enhances the pro-survival Wnt and Akt signaling pathways in tumor cells to prevent stress-induced apoptosis and increase the number of CSCs for promotion of early metastatic colonization of tissues. Importantly, the production of POSTN is predominately induced by TGF-?, a molecule that is up regulated in the tumor environment following exposure to chemotherapy. Based on these observations, we hypothesize that POSTN production in response to chemotherapy induces a protective, pro-survival environment within established metastases through induction of a CSC phenotype via activation of Wnt and\or Akt signaling pathways, and that POSTN production is regulated primarily by TGF-? from tumor-associated macrophages (TAMs). To answer these questions, we propose 3 specific aims, which we anticipate will fill a critical knowledge gap regarding therapy-induced changes within the metastatic tumor environment, revealing a significant role for POSTN in mediating acquired drug resistance in the metastatic setting. In Aim 1, we will use organotypic lung-like cultures to identify the cytotoxic drug(s) that most strongly induce POSTN-mediated chemoresistance in human breast cancer cells. We will determine if this resistance is mediated through activation of the Wnt and\or Akt signaling pathways and induction of a CSC phenotype using western blot and flow cytometry. For aim 2, we will use mouse models of breast cancer metastasis to identify the key cytokines and their cellular sources regulating POSTN production within established metastases following chemotherapy. This will be accomplished via flow cytometry, fluorescence microscopy, and RT-qPCR, and confirmed by in vivo antibody neutralization of these cytokines in the mouse models. Lastly, in aim 3, we will determine the effects of macrophage depletion on drug-induced POSTN production within established pulmonary metastases using transgenic mice and small molecule drugs, which deplete tumor-associated macrophages. To fully elucidate the potential for interruption of POSTN signaling in res-establishment of chemosensitivity, established metastatic tumor growth responses to chemotherapy alone, or chemotherapy in combination with either macrophage depletion or neutralization of POSTN or TGF-? will be directly compared using survival studies, bioluminescence imaging, and histology in mouse models of metastasis.